69-tetrahydrocannabinol (THC), the psychoactive component of marijuana, produces a variety of effects by activating cannaibinoid CBl receptors (CB1R) in specific CNS regions. THC is widely used illicitly for its psychoactive properties, which can lead to addiction, and is the primary component of antiemetic, appetite stimulating and anti-spasticity medications. Chronic THC use produces tolerance and dependence, which might contribute to addiction and limit the therapeutic use of cannabinoids. Our previous studies revealed brain region-specific CB1R desensitization and downregulation after THC administration, and this receptor neuroadaptation was associated with tolerance to in vivo effects. The proposed studies will elucidate the role of specific signaling proteins, J3-arrestin and 6FosB in regulation of acute and chronic CB1R signaling using mouse models with abrogated or overexpressed proteins. CBl R function will be assessed by functional neuroanatomy and biochemical pharmacology. Parallel in vivo studies will determine whether altered CB1R signaling translates into behavioral and physiological effects. Specific Aim 1 will assess CB1R function and in vivo effects in wild-type and J3-arrestin-2 null mice to test the hypothesis that J3-arrestin regulates acute and chronic CB1R signaling, THC-mediated in vivo effects (antinociception, locomotor inhibition, catalepsy, hypothermia) and tolerance. Preliminary data show that CB1R-mediated G-protein activity is increased in na'ive J3-arrestin-2 null mouse spinal cord and CBl R desensitization following chronic THC is reduced in 13-arrestin-2 null mice. Previous studies showed that CBl R desensitization and down regulation recover by 2 weeks following chronic THC treatment and therefore lack the stability to underlie persistent changes associated with addiction. Specific Aim 2 hypothesizes that 6FosB, a stable transcription factor, is involved in chronic THC-mediated effects. The regional expression of 6FosB after THC treatment will first be examined. Then CB1R signaling and THC in vivo effects will be assessed in transgenic mice with inducible overexpression or dominant negative inactivation of 6FosB in the striatum. Preliminary studies show increased 6FosB in nucleus accumbens following chronic THC treatment, and administration of THC to 6FosB overexpressing mice revealed attenuated tolerance and CB1R desensitization in mice. These studies will elucidate cellular and molecular mechanisms that regulate CBl R signaling and neuroadaptation and translate these effects to the in vivo level. This project will determine roles of specific signaling proteins in the effects of acute and long-term CB1R activation by THC and are relevant to cannabinoid therapeutics and drug abuse.